DNA REPLICATION PROTEIN CDC6 REGULATION AND FUNCTION

DNA 复制蛋白 CDC6 调节和功能

• 批准号: 6525503
• 负责人: THOMAS R COLEMAN
• 金额: $ 5.52万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2002-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6525503
• 关键词: DNA replication; DNA replication origin; Xenopus oocyte; cell cycle proteins; cell differentiation; cell growth regulation; enzyme activity; fluorescence microscopy; immunoaffinity chromatography; immunofluorescence technique; laboratory rabbit; mass spectrometry; microinjections; nuclear membrane; phosphorylation; protein binding; protein structure function; western blottings

A fundamental control point governing growth and differentiation is the faithful duplication of the eukaryotic genome. Genetic studies in budding yeast have identified several proteins that assemble into a "pre-initiation complex" at specific chromosome locations during G1 phase of the cell cycle. Cdc6 homologues are essential components of this complex; they are necessary for DNA initiation, are modified in a cell cycle-dependent manner, and their destruction may function in the block to re-replication. I will investigate the biochemical basis of DNA replication using Xenopus egg extracts. The Xenopus egg extract supports efficient DNA replication, cell cycle progression, and nuclear envelope formation and breakdown in vitro. Using the Xenopus extract, we identified and characterized the Xenopus Cdc6 (Xcdc6) protein which, like its yeast homologues, plays a critical early step in chromosomal replication. We demonstrated that it is essential for initiation of DNA replication, coordinates the binding of other replication machinery, and its relocalization to the nuclear envelope may serve to prevent re- initiation. Using the Xenopus extract model system, I will address two major questions: 1)how does Xdc6 contribute to the initiation of DNA replication and 2)what mechanisms regulate the Xcdc6 activity? To this end, I will: 1)Map the functional domains of Xcdc6. I will address structure/function issues by characterizing various truncated and mutant forms of Xcdc6 to evaluate their activity and localization. 2)Assess the role of phosphorylation on Xcdc6 function. Xcdc6 is phosphorylated in a cell cycle-dependent manner. To investigate whether phosphorylation regulates Xcdc6 function, I will define the residues modified and evaluate the contribution of these modifications to Xcdc6 regulation and localization by characterizing appropriate point mutants. Given that Xenopus and human Cdc6 are 80 percent identical, what we learn in biochemically-tractable frog extract systems will advance our understanding of human cell-cycle control. Information derived from these studies should suggest effective strategies to combat uncontrolled cell division, which is the hallmark of cancer.

控制生长和分化的基本控制点是 真核生物基因组的忠实复制。 遗传研究 芽殖酵母已经鉴定出几种蛋白质， G1期特定染色体位置的“前起始复合体” 细胞周期的一个阶段。 Cdc 6同源物是 这种复合物;它们是DNA起始所必需的， 细胞周期依赖的方式，它们的破坏可能在细胞周期中起作用。 块重新复制。 我将研究 使用爪蟾卵提取物进行DNA复制。 非洲爪蟾卵提取物 支持有效的DNA复制、细胞周期进程和细胞核 体外包膜形成和破坏。 使用非洲爪蟾提取物， 我们鉴定并表征了非洲爪蟾Cdc 6（Xcdc 6）蛋白， 像它的酵母同源物一样，在染色体上起着关键的早期作用。 复制的 我们证明了它对DNA的起始是必不可少的， 复制，协调其他复制机器的结合， 它的重新定位到核膜可能有助于防止重新定位， 入会仪式 使用非洲爪蟾提取模型系统，我将解决两个主要问题， 问题：1）Xdc 6如何促进DNA的启动 2）什么机制调节Xcdc 6活性？本 最后，我将：1）定位Xcdc 6的功能域。 我将向 通过表征各种截短和突变体的结构/功能问题 Xcdc 6的形式，以评估其活性和定位。 2）评估 磷酸化对Xcdc 6功能的作用。 Xcdc 6被磷酸化 以细胞周期依赖的方式。 调查是否 磷酸化调节Xcdc 6功能，我将定义残基 修改并评估这些修改对Xcdc 6的贡献 通过表征适当的点突变体来调节和定位。 考虑到非洲爪蟾和人类Cdc 6有80%的相同性， 在生物化学上易处理青蛙提取系统中的学习将推进我们的 了解人类细胞周期控制。导出的信息 这些研究应该提出有效的策略， 细胞分裂这是癌症的标志